As reported last year, the active site cysteine of peroxiredoxins is selectively oxidized to cysteine sulfinic acid (Cys!VSO2H) during catalysis, resulting in inactivation of peroxidase activity. Metabolic labeling of mammalian cells with 35S has now revealed that the sulfinic form of peroxiredoxin I produced during exposure of cells to H2O2 is rapidly reduced to the catalytically active thiol form. This ability of mammalian cells to reduce protein sulfinic acid is contrary to the general belief that oxidation to the sulfinic state is an irreversible process in cells. This reduction reaction might serve as a mechanism to repair oxidatively damaged proteins or represent a new type of cyclic modification by which the function of various proteins is regulated. We have identified and characterized a14-kDa human thioredoxin (Trx)-related protein designated TRP14. This cytosolic protein was expressed in all tissues and cell types examined, generally in smaller amounts than Trx1. Biochemical characterization of TRP14 suggested that, like Trx1, TRP14 is a disulfide reductase; its active site cysteine is sufficiently nucleophilic and its redox potential is similar to those of other cellular thiol reductants. However, although TRP14 reduced small disulfide-containing peptides, it did not reduce the disulfides of several Trx1 substrates, suggesting that TRP14 and Trx1 interact with distinct targets. The possible role of TRP14 in tumor necrosis factor-alpha (TNF-alpha) signaling was also studied in comparison with Trx1 by RNA interference in HeLa cells. Partial depletion of TRP14 augmented the TNF-alpha- induced phosphorylation and degradation of IkBalpha as well as the consequent activation of NF-kB to a greater extent than did Trx1 depletion. Deficiency of TRP14 or Trx1 enhanced TNF-alpha induced activation of caspases and subsequent apoptosis by similar extents. The TNF-alpha-induced activation of JNK and p38 mitogen-activated protein kinases, however, was promoted by depletion of TRP14 but not by that of Trx1. Unlike Trx1, TRP14 neither associated with nor inhibited the kinase activity of ASK1. These results suggest that TRP14 modulates TNF-alpha signaling pathways, probably by interacting with proteins distinct from the targets of Trx1. Tumor necrosis factor-alpha (TNF-alpha) induces the activation of all three types of mitogen-activated protein kinase (MAPK): c-Jun NH2-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK). This cytokine also induces the production of several types of reactive oxygen species including H2O2. With the use both of HeLa cells expressing wild-type or dominant negative forms of the cytosolic peroxidase peroxiredoxin II and of mouse embryonic fibroblasts deficient in this protein, we evaluated the roles of H2O2 in the activation of MAPKs by TNF-alpha. In vitro kinase assays as well as immunoblot analysis with antibodies specific for activated MAPKs indicated that H2O2 produced in response to TNF-alpha potentiates the activation of JNK and p38 induced by this cytokine but inhibits that of ERK. Our results also suggest that cytosolic peroxiredoxins are important regulators of TNF signaling pathways.